Lipopolysaccharides (lps)

To investigate the role of AT-EVs in inflammation, 5 × 10⁵ RAW 264.7 macrophages were seeded in six-well plates and cultured with high-glucose DMEM with FBS, until cells reached 80% confluence. As previously described (Xu et al., 2020 (link)), cells were divided into five groups for which the culture medium was replaced as follows: Control group: cells were cultured with DMEM; Lipopolysaccharide (LPS) + IFNγ group: cells were cultured with DMEM +1 μg/mL LPS (#L2880, Sigma-Aldrich, St. Louis, MO, United States) + 30 ng/mL IFNγ (#315-05-100, PeproTech, Rocky Hill, NJ, United States); LPS + IFNγ + dAT-EVs group: cells were cultured with 1 μg/mL LPS +30 ng/mL IFNγ +150 μg/mL dAT-EVs; LPS + IFNγ + mAT-EVs group: cells were cultured with 1 μg/mL LPS +30 ng/mL IFNγ +150 μg/mL mAT-EVs; LPS + IFNγ + cAT-EVs group: cells were cultured with 1 μg/mL LPS +30 ng/mL IFNγ +150 μg/mL cAT-EVs. After induction for 24 h, 2 × 10⁵ cells were incubated with fluorescein isothiocyanate (FITC)-anti-mouse CD86 (1:50, #105110, BioLegend, San Diego, CA, United States) and PE-anti-mouse CD206 (1:40, #141706, BioLegend) at 4°C for 30 min, before being washed twice with staining buffer (#00-4222-26, Invitrogen, San Diego, CA, United States of America). Polarization of RAW 264.7 cells was determined using flow cytometry (BD FACSCalibur, Beckman Coulter, Inc., Brea, CA, United States).

The anti-inflammatory potential of the peptide fraction 2–10 kDa was examined in coN cells with few alterations from what was previously published [78 (link)]. Fibroblasts and coN cells were seeded in 25 cm² T-flasks and then incubated for 24 h at 37 °C, 5% (v/v) CO₂ and 99% (v/v) relative humidity. Cells were incubated for 2 h with 7 µg/mL lipopolysaccharide (LPS, Sigma Aldrich, St. Louis, MO, USA), then 0.25 mg/mL peptide fraction 2–10 kDa or 1% (v/v) ethanol were added, and cells incubated for further 3 h. Similar samples without LPS were also prepared in parallel. Samples treated and untreated with LPS (+LPS and −LPS, respectively) were collected after 2 h incubation with LPS, and +LPS and −LPS samples incubated with peptide fraction or respective negative control were collected after 3 h (5 h since the beginning of the experiment) by cell detachment with Tryple Express (ThermoFisher Scientific), centrifugation at 500× g and pellet resuspension in NZYol (NZYtech, Lisbon, Portugal). RNA was extracted according to manufacturer’s instructions, cDNA synthesized using the NZY M-MULV First strand cDNA synthesis kit (NZYtech), and TNF-α and RNA 18S genes were amplified using the NZY Supreme qPCR Green Master Mix (NZYtech) in a Corbett-Rotor Gene thermal cycler (Qiagen, Hilden, Germany). The expression of TNF-α in samples was determined with the 2^−∆∆Ct method [79 (link)].

The 16HBE cells were treated with lipopolysaccharide (LPS) (1 mg/L, 00‐4976‐03, Invitrogen) at 37°C for 24 h, and then cultured with 15 or 30 μM Pts at 37°C for 24 h. Accordingly, Control group (without drug treatment), LPS group (LPS), 15 μM Pts (LPS + Pts15) group, and 30 μM Pts (LPS + Pts30) group were set up. For protein inhibition, 16HBE cells were first treated with Compound C (AMPK inhibitor, Cat: HY‐13418A; MCE), EX‐527 (Sirt1 inhibitor, Cat: HY‐15452; MCE), or ML385 (Nrf2 inhibitor, Cat: HY‐100523; MCE) for 2 h and then with Pts (30 mg/kg) for 1 h. For small interfering RNA transfection, 16HBE cells were transfected with siControl (#6568; Cell Signaling Technology), siAMPK (#6620; Cell Signaling Technology), siSirt1 (#12241; Cell Signaling Technology) and siNrf2 (#5285; Cell Signaling Technology) groups with Lipofectamine 2000 (#11668027; Invitrogen). After 6 h, fresh DMEM was added to continue the culture for 48 h.